Celery named ADS-3

ABSTRACT

A novel celery cultivar, designated ADS-3, is disclosed. The invention relates to the seeds of celery cultivar ADS-3, to the plants of celery line ADS-3 and to methods for producing a celery plant by crossing the cultivar ADS-3 with itself or another celery line. The invention further relates to methods for producing a celery plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other celery lines derived from the cultivar ADS-3.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive celery (Apiumgraveolens var. dulce) variety, designated ADS-3. There are numeroussteps in the development of any novel, desirable plant germplasm. Plantbreeding begins with the analysis and definition of problems andweaknesses of the current germplasm, the establishment of program goals,and the definition of specific breeding objectives. The next step isselection of germplasm that possess the traits to meet the programgoals. The goal is to combine in a single variety or hybrid an improvedcombination of desirable traits from the parental germplasm. Theseimportant traits may include increased stalk size and weight, higherseed yield, improved color, resistance to diseases and insects,tolerance to drought and heat, and better agronomic quality.

Practically speaking, all cultivated forms of celery belong to thespecies Apium graveolens var. dulce that is grown for its edible stalk.As a crop, celery is grown commercially wherever environmentalconditions permit the production of an economically viable yield. In theUnited States, the principal growing regions are California, Florida,Texas and Michigan. Fresh celery is available in the United Statesyear-round although the greatest supply is from November throughJanuary. For planting purposes, the celery season is typically dividedinto two seasons, summer and winter, with Florida, Texas and thesouthern California areas harvesting from November to July, and Michiganand northern California harvesting from July to October. Fresh celery isconsumed as fresh, raw product and occasionally as a cooked vegetable.

Celery is a cool-season biennial that grows best from 600 to 65° F. (160to 18° C.), but will tolerate temperatures from 45° to 75° F. (70 to 24°C.). Freezing will damage mature celery by splitting the petioles orcausing the skin to peal, making the stalks unmarketable. This is anoccasional problem in plantings in the winter regions. However, celerycan tolerate minor freezes early in the crop.

The two main growing regions for celery (Apium graveolens L.) inCalifornia are located along the Pacific Ocean: the central coast orsummer production area (Monterey, San Benito, Santa Cruz and San LuisObispo Counties) and the south coast or winter production area (Venturaand Santa Barbara Counties). A minor region (winter) is located in thesouthern deserts (Riverside and Imperial Counties).

In the south coast, celery is transplanted from early August to Aprilfor harvest from November to mid-July; in the Santa Maria area, celeryis transplantaed from January to August for harvest from April throughDecember. In the central coast, fields are transplanted from March toSeptember for harvest from late June to late December. In the southerndeserts, fields are transplanted in late August for harvest in January.

Commonly used celery varieties for coastal production include Tall Utah52-75, Conquistador and Sonora. Some shippers use their own proprietaryvarieties. Celery seed is very small and difficult to germinate. Allcommercial celery is planted as greenhouse-grown transplants. Celerygrown from transplants is more uniform than from seed and takes lesstime to grow the crop in the field. Transplanted celery is placed indouble rows on 40-inch (100-cm) beds with plants spaced between 6.7 and7 inches (22.5-cm) apart.

Celery is an allogamous biennial crop. Celery consists of 11chromosomes. Its high degree of out-crossing is accomplished by insectsand wind pollination. Pollinators visiting celery flowers include alarge number of wasp, bee and fly species. Celery is subject toinbreeding depression, which appears to be genotype dependent, sincesome lines are able to withstand continuous selfing for three or fourgenerations. Crossing of inbreds results in heterotic hybrids that arevigorous and taller than sib-mated or inbred lines.

Celery flowers are protandrous, with pollen being released 3-6 daysbefore stigma receptivity. At the time of stigma receptivity the stamenswill have fallen and the two stigmata unfolded in an upright position.The degree of protandy varies, which makes it difficult to performreliable hybridization, due to the possibility of accidental selfing.

Celery flowers are very small, significantly precluding easy removal ofindividual anthers. Furthermore, different developmental stages of theflowers in umbels makes it difficult to avoid uncontrolled pollinations.The standard hybridization technique in celery consists of selectingflower buds of the same size and eliminating the older and youngerflowers. Then, the umbellets are covered with glycine paper bags for a5-10 day period, during which the stigmas become receptive. At the timethe flowers are receptive, available pollen or umbellets shedding pollenfrom selected male parents are rubbed on to the stigmas of the femaleparent.

Plants require a period of vernalization while in the vegetative phasein order to induce seed stalk development. A period of 6-10 weeks at5-8° C. is usually adequate. However, unless plants are beyond ajuvenile state or a minimum of 4 weeks old they may not be receptive tovernalization. Due to a wide range of response to the cold treatment, itis often difficult to synchronize crossing, since plants will flower atdifferent times. However, pollen can be stored for 6-8 months at −10° C.in the presence of silica gel or calcium chloride with a viabilitydecline of only 20-40%, thus providing flexibility to perform crossesover a longer time.

For selfing, the plant or selected umbels are caged in cloth bags. Theseare shaken several times during the day to promote pollen release.Houseflies (Musca domestics) can also be introduced weekly into the bagsto perform pollinations.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three years at least. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits areused as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from ten to twenty years from the time thefirst cross or selection is made. Therefore, development of newcultivars is a time-consuming process that requires precise forwardplanning, efficient use of resources, and a minimum of changes indirection.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of plant breeding is to develop new, unique and superior celerycultivars. The breeder initially selects and crosses two or moreparental lines, followed by repeated selfing and selection, producingmany new genetic combinations. The breeder can theoretically generatebillions of different genetic combinations via crossing, selfing andmutations. The breeder has no direct control at the cellular level.Therefore, two breeders will never develop the same line, or even verysimilar lines, having the same celery traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The cultivarsthat are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large research monies todevelop superior celery cultivars.

The development of commercial celery cultivars requires the developmentof celery varieties, the crossing of these varieties, and the evaluationof the crosses. Pedigree breeding and recurrent selection breedingmethods are used to develop cultivars from breeding populations.Breeding programs combine desirable traits from two or more varieties orvarious broad-based sources into breeding pools from which cultivars aredeveloped by selfing and selection of desired phenotypes. The newcultivars are crossed with other varieties and the hybrids from thesecrosses are evaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits, are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self- or cross-pollinating crops. A genetically variablepopulation of heterozygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror line that is the recurrent parent. The source of the trait to betransferred is called the donor parent. The resulting plant is expectedto have the attributes of the recurrent parent (e.g., cultivar) and thedesirable trait transferred from the donor parent. After the initialcross, individuals possessing the phenotype of the donor parent areselected and repeatedly crossed (backcrossed) to the recurrent parent.The resulting plant is expected to have the attributes of the recurrentparent (e.g., cultivar) and the desirable trait transferred from thedonor parent.

The single-seed descent procedure in the strict sense refers to plantinga segregating population, harvesting a sample of one seed per plant, andusing the one-seed sample to plant the next generation. When thepopulation has been advanced from the F₂ to the desired level ofinbreeding, the plants from which lines are derived will each trace todifferent F₂ individuals. The number of plants in a population declineseach generation due to failure of some seeds to germinate or some plantsto produce at least one seed. As a result, not all of the F₂ plantsoriginally sampled in the population, will be represented by a progenywhen generation advance is completed

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., “Principles of Plant Breeding” John Wiley and Son, pp.115-161, 1960; Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987; “Carrots and Related Vegetable Umbelliferae”, Rubatzky, V. E., etal., 1999).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Celery in general is an important and valuable vegetable crop. Thus, acontinuing goal of plant breeders is to develop stable, high yieldingcelery cultivars that are agronomically sound. The reasons for this goalare obviously to maximize the amount of yield produced on the land. Toaccomplish this goal, the celery breeder must select and develop celeryplants that have the traits that result in superior cultivars.

BRIEF DESCRIPTION OF THE INVENTION

According to the invention, there is provided a novel celery cultivar,designated ADS-3. This invention thus relates to the seeds of celerycultivar ADS-3, to the plants of celery cultivar ADS-3 and to methodsfor producing a celery plant produced by crossing the celery ADS-3 withitself or another celery line, and to methods for producing a celeryplant containing in its genetic material one or more transgenes and tothe transgenic celery plants produced by that method. This inventionalso relates to methods for producing other celery cultivars derivedfrom celery cultivar ADS-3 and to the celery cultivar derived by the useof those methods. This invention further relates to hybrid celery seedsand plants produced by crossing the line ADS-3 with another celery line.

In another aspect, the present invention provides regenerable cells foruse in tissue culture of celery cultivar ADS-3. The tissue culture willpreferably be capable of regenerating plants having the physiologicaland morphological characteristics of the foregoing celery plant, and ofregenerating plants having substantially the same genotype as theforegoing celery plant. Preferably, the regenerable cells in such tissuecultures will be embryos, protoplasts, seeds, callus, pollen, leaves,anthers, roots, suckers and meristematic cells. Still further, thepresent invention provides celery plants regenerated from the tissuecultures of the invention.

Another objective of the invention is to provide methods for producingother celery plants derived from celery cultivar ADS-3. Celery cultivarsderived by the use of those methods are also part of the invention.

The invention also relates to methods for producing a celery plantcontaining in its genetic material one or more transgenes and to thetransgenic celery plant produced by that method.

In another aspect, the present invention provides for single geneconverted plants of ADS-3. The single transferred gene may preferably bea dominant or recessive allele. Preferably, the single transferred genewill confer such trait as male sterility, herbicide resistance, insectresistance, resistance for bacterial, fungal, or viral disease, malefertility, enhanced nutritional quality and industrial usage. The singlegene may be a naturally occurring celery gene or a transgene introducedthrough genetic engineering techniques.

The invention further provides methods for developing celery plant in acelery plant breeding program using plant breeding technique includingrecurrent selection, backcrossing, pedigree breeding, restrictionfragment length polymorphism enhanced selection, genetic marker enhancedselection and transformation. Seeds, celery plant, and parties thereofproduced by such breeding methods are also part of the invention.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Allele. The allele is any of one or more alternative form of a gene, allof which alleles relates to one trait or characteristic. In a diploidcell or organism, the two alleles of a given gene occupy correspondingloci on a pair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics, except for the characteristics derived from theconverted gene.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Single gene converted. Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of a line are recovered in addition to thesingle gene transferred into the line via the backcrossing technique orvia genetic engineering.

Maturity Date. Maturity in celery can be dictated by two conditions. Thefirst, or true maturity, is the point in time when the celery reachesmaximum size distribution, but before defects such as pith, yellowing,feather-leaf or brownstem appear. The second, or market maturity is anartificial maturity dictated by market conditions, i.e, the marketrequirement may be for 3 dozen sizes so the field is harvested atslightly below maximum yield potential because the smaller sizes arewhat the customers prefer at that moment.

RHS. RHS refers to the Royal Horticultural Society of England whichpublishes an official botanical color chart quantitatively identifyingcolors according to a defined numbering system, The chart may bepurchased from Royal Hort Society Enterprise Ltd RHS Garden; Wisley,Woking; Surrey GU236QB, UK.

Celery Yield (Tons/Acre). The yield in tons/acre is the actual yield ofthe celery at harvest.

Theoretical Maximum Yield. If you assume 100% 2 dozen size and a 35,000plant population per acre and 60 pound cartons, your theoretical maximumyield would be 43.7 tons.

Pith. Pith is a sponginess/hollowness/white discoloration that occurs inthe petioles of varieties naturally as they become over mature. In somevarieties it occurs at an earlier stage causing harvest to occur priorto ideal maturity. Pith generally occurs in the outer older petiolesfirst. If it occurs, these petioles are stripped off to make grade andeffectively decreases the stalk size and overall yield potential.

Suckers. Suckers are auxiliary shoots that form at the base of the stalkor within the auxiliary buds between each petiole. If these shoots formbetween the petioles of the stalk, several petioles have to be strippedoff causing the celery to become smaller and the functional yields to bedecreased.

Feather leaf. Feather leaf is a yellowing of the lower leaves. Itgenerally occurs in the outer petioles but can also be found on innerpetioles of the stalk. These yellowing leaves which would normallyremain in the harvested stalk are considered unacceptable. Thesepetioles then have to be stripped off in order to meet market gradewhich effectively decreases the stalk size and yield.

Black heart. Black heart is due to a lack of movement of sufficientcalcium that causes the plant to turn brown and begin to decay at thegrowing point of the plant. Celery, in certain conditions such as warmweather, grows very rapidly and incapable of moving sufficient amountsof calcium to the growing point.

DETAILED DESCRIPTION OF THE INVENTION

ADS-3 celery variety is the first of a new class of celery for Florida.Developed as a processor, specifically for mechanical harvest andprocessing, it has 2′-5 additional petioles compared to most Floridavarieties and is 5-10 cm longer to the joint. The petioles are mediumwidth, but unlike current varieties with fairly large wings. ADS-3'swidth does not change from the butt to the joint. The variety is darkgreen and consistent for petiole width from the inner petioles to theoutside petioles. These characteristics maximize the processing qualityfor all segments of the processing trade (3-4 inch sticks, crescents anddices).

ADS-3 is tolerant to Pseudomonas apii and Fusarium oxysporum f. v. apiirace 2 moderately tolerant to pith and Cercospora apii.

ADS-3 is similar to Camlynn variety which it resembles only in overallplant height and length to the joint. ADS-3 has both, larger petioles(width) and greater quantity of petioles. Camlynn has little to nodisease tolerance to Pseudomonas apii, Fusarium oxysporum f. v. apiirace 2 and Cercospora apii.

Some of the criteria used to select in various generations include:color, disease resistances, stalk weight, number of leaves, appearanceand length, yield, emergence, maturity, plant architecture, seed yieldand quality, and disease resistance

The cultivar has shown uniformity and stability for the traits, withinthe limits of environmental influence for the traits. It has beenself-pollinated a sufficient number of generations with carefulattention to uniformity of plant type. The line has been increased withcontinued observation for uniformity. No variant traits have beenobserved or are expected in ADS-3.

Celery cultivar ADS-3 has the following morphologic and othercharacteristics (based primarily on data collected at Belle Glade,Florida Research Station and field plots). TABLE 1 VARIETY DESCRIPTIONINFORMATION Maturity: 90 days in Eastern U.S. Plant Height: 95 cm Numberof Outer Petioles (>40 cm): 12 Number of Inner Petioles (<40 cm): 5Stalk Shape: Cylindrical Stalk Conformation: Compact Heart Formation:Medium Petiole Length 36 cm (from butt to first joint): Petiole LengthClass: Long (>30 cm) Petiole Width (at midpoint) 21 mm Petiole Thickness(at midpoint) 11 mm Cross Section Shape: Deeply Cupped Color (unblanchedat harvest) Dark Green - 5GY 4/6 Munsell Color Anthocyanin: AbsentStringiness: Normal Ribbing: Moderate Glossiness: Moderately Glossy LeafBlade Color: Dark Green - 5GY 3/4 Bolting: Fairly Suscepticle StressTolerance: Adaxial Crackstem (Boron Deficiency) - Tolerant AbaxialCrackstem (Boron Deficiency) - Tolerant Leaf Margin Chlorosis (MagnesiumDeficiency) - Tolerant Blackheart (Calcium Deficiency) - TolerantPithiness (Nutritional Deficiency) - Moderate Tolerance Feather Leaf -Tolerant Sucker Development - Tolerant Disease Resistance: SouthernBacteria Blight (Psuedomonus chichorii) - Tolerant Early Blight(Cercospora apii) - Slight Tolerance Fusarium Yellows, Race 2 (Fusariumoxysporum) - Moderate Tolerance Brown Stem - Tolerant

Tables

In the tables that follow, the traits and characteristics of celerycultivar ADS-3 are given compared to other check cultivars.

Table 2 shows trait comparisons between ADS-3 and Florida 683 ‘K’Strain, Florida Slowbolt, Junebelle and Floribelle under normal Floridaconditions. Rating scale for Southern Bacterial Blight (Psuedomonuschichorii) and Early Blight (Cercospora apii) is 0 to 10, 0 being freeof disease and 10 most severely infected.

Table 3 shows yield comparisons between ADS-3 and Florida 683 ‘K’Strain. Both varieties were mechanically harvested in Belle Glade,Florida and then processed to produce ½ inch crescents. The mechanicalharvestor removes the tops and butts of the celery stalk in the field.All that is taken to the processing plant are the celery limbs. As shownin this table, ADS-3 has more limb and ½ inch crescent yield as comparedto Florida 683 ‘K’ Strain. The table also shows that the crescent yieldis due to the additional limb material available to process in ADS-3.ADS-3 is more uniform in crescent size, color, grade, and quality thanFlorida 683 ‘K’ strain. TABLE 2 Comparisons with other varieties undernormal conditions Florida 683 FL ADS-3 ‘K’ Strain Slowbolt JunebelleFloribelle LSD .05 Plant Height (cm) 88.7 78.8 77.7 68.5 69.6 2.37Number of Outer Petioles 11.9 9.8 9.1 9.3 9.2 1.54 Number of InnerPetioles 6.0 6.7 5.6 7.8 7.1 0.93 Length of Outer Petioles @ midrib (cm)38.0 24.4 30.0 23.8 26.6 1.53 Width of Outer Petioles @ midrib (mm) 15.920.7 20.0 18.8 19.6 0.90 Thickness of Outer Petioles @ midrib (mm) 7.79.9 9.6 8.7 9.6 0.63 Southern Bacterial Blight 0 6 6 7 3 Early Blight 37 0 0 0

TABLE 3 Comparison with Florida 683 ‘K’ Strain for processing yieldFlorida 683 ADS-3 ‘K’ Strain Limb yield per acre (pounds) 62,127 55,179Unuseable product (pounds)  1,053   980 Yield of ½ inch crescents(pounds) 61,087 54,199 Yield efficiency (percent)     98%     98%

FURTHER EMBODIMENTS OF THE INVENTION

This invention also is directed to methods for producing a celerycultivar plant by crossing a first parent celery plant with a secondparent celery plant wherein either the first or second parent celeryplant is a celery plant of the line ADS-3. Further, both first andsecond parent celery plants can come from the cultivar ADS-3. Stillfurther, this invention also is directed to methods for producing acultivar ADS-3-derived celery plant by crossing cultivar ADS-3 with asecond celery plant and growing the progeny seed, and repeating thecrossing and growing steps with the cultivar ADS-3-derived plant from 0to 7 times. Thus, any such methods using the cultivar ADS-3 are part ofthis invention: selfing, backcrosses, hybrid production, crosses topopulations, and the like. All plants produced using cultivar ADS-3 as aparent are within the scope of this invention, including plants derivedfrom cultivar ADS-3. Advantageously, the cultivar is used in crosseswith other, different, cultivars to produce first generation (F₁) celeryseeds and plants with superior characteristics.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which celery plants can be regenerated,plant calli, plant clumps and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, seeds, roots,anthers, suckers and the like.

As is well known in the art, tissue culture of celery can be used forthe in vitro regeneration of a celery plant. Tissue culture of varioustissues of celerys and regeneration of plants therefrom is well knownand widely published. For example, reference may be had to Teng et al.,HortScience. 1992, 27: 9, 1030-1032 Teng et al., HortScience. 1993, 28:6, 669-1671, Zhang et al., Journal of Genetics and Breeding. 1992, 46:3, 287-290, Webb et al., Plant Cell Tissue and Organ Culture. 1994, 38:1, 77-79, Curtis et al., Journal of Experimental Botany. 1994, 45: 279,1441-1449, Nagata et al., Journal for the American Society forHorticultural Science. 2000, 125: 6, 669-672. It is clear from theliterature that the state of the art is such that these methods ofobtaining plants are, and were, “conventional” in the sense that theyare routinely used and have a very high rate of success. Thus, anotheraspect of this invention is to provide cells which upon growth anddifferentiation produce celery plants having the physiological andmorphological characteristics of variety ADS-3.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed line.

Plant transformation involves the construction of an expression vectorthat will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed celery plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the celery plant(s).

Expression Vectors for celery Transformation

Marker Genes—Expression vectors include at least one genetic marker,operably linked to a regulatory element (a promoter, for example) thatallows transformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase ll (nptll) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990<Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include â-glucuronidase (GUS, â-galactosidase,luciferase and chloramphenicol, acetyltransferase. Jefferson, R. A.,Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989),Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock etal., EMBO J. 3:1681 (1984).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes publication 2908, Imagene Green, p. 1-4 (1993) and Naleway etal., J. Cell Biol. 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orscierenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression incelery. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in celery. With an inducible promoter the rateof transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression incelery or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in celery.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last etal., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3):291-300 (1992)).

The ALS promoter, Xba1/NcoI fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/NcoIfragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin celery. Optionally, the tissue-specific promoter is operably linkedto a nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in celery. Plants transformed with agene of interest operably linked to a tissue-specific promoter producethe protein product of the transgene exclusively, or preferentially, ina specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondroin or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell.Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is celery. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes That Confer Resistance to Pests or Disease and That Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. Tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btα-endotoxin gene. Moreover, DNA molecules encoding a-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

C. A lectin. See, for example, the disclose by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus á-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper accumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung celery calmodulin cDNA clones, and Griesset al., Plant Physiol. 104:1467 (1994), who provide the nucleotidesequence of a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-α, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo α-1,4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa celery endopolygalacturonase-inhibiting protein is described byToubart et al., Plant J. 2:367 (1992).

R. A development-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bio/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

R. A lettuce mosaic potyvirus (LMV) coat protein gene introduced intoLactuca Sativa in order to increase its resistance to LMV infection. SeeDinant et al., Molecular Breeding. 1997, 3: 1, 75-86.

2. Genes That Confer Resistance to a Herbicide, For Example:

A. A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl. Genet. 80:449(1990), respectively.

B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy propionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. Seealso Umaballava-Mobapathie in Transgenic Research. 1999, 8: 1, 33-44that discloses lactuca sativa resistant to glufosinate. European patentapplication No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374to Goodman et al., disclose nucleotide sequences of glutamine synthetasegenes which confer resistance to herbicides such as L-phosphinothricin.The nucleotide sequence of a phosphinothricin-acetyl-transferase gene isprovided in European application No. 0 242 246 to Leemans et al.,DeGreef et al., Bio/Technology 7:61 (1989), describe the production oftransgenic plants that express chimeric bar genes coding forphosphinothricin acetyl transferase activity. Exemplary of genesconferring resistance to phenoxy propionic acids and cycloshexones, suchas sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genesdescribed by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

C. A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes That Confer or Contribute to a Value-Added Trait, Such as:

A. Increased iron content of the celery, for example by transforming aplant with a soybean ferritin gene as decribed in Goto et al., ActaHorticulturae. 2000, 521, 101-109. Parallel to the improved iron contentenhanced growth of transgenic celerys was also observed in earlydevelopment stages.

B. Decreased nitrate content of leaves, for example by transforming acelery with a gene coding for a nitrate reductase. See for exampleCurtis et al., Plant Cell Report. 1999, 18: 11, 889-896.

C. Increased sweetness of the celery by transferring a gene coding formonellin, that elicits a flavor 100000 times sweeter than sugar on amolar basis. See Penarrubia et al., Biotechnology. 1992, 10: 5, 561-564.

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985). Curtis et al., Journal ofExperimental Botany. 1994, 45: 279, 1441-1449, Torres et al., Plant cellTissue and Organic Culture. 1993, 34: 3, 279-285, Dinant et al.,Molecular Breeding. 1997, 3: 1, 75-86. A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal or vegetable crop species andgymnosperms have generally been recalcitrant to this mode of genetransfer, even though some success has recently been achieved in riceand corn. Hiei et al., The Plant Journal 6:271-282 (1994) and U.S. Pat.No. 5,591,616 issued Jan. 7, 1997. Several methods of planttransformation, collectively referred to as direct gene transfer, havebeen developed as an alternative to Agrobacterium-mediatedtransformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 im. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Russell, D. R.,et al. PI. Cell. Rep. 12(3, January), 165-169 (1993), Aragao, F. J. L.,et al. Plant Mol. Biol. 20(2, October), 357-359 (1992), Aragao, F. J.L., et al. PI. Cell. Rep. 12(9, July), 483-490 (1993). Aragao Theor.Appl. Genet. 93: 142-150 (1996), Kim, J.; Minamikawa, T. Plant Science117: 131-138 (1996), Sanford et al., Part. Sci. Technol. 5:27 (1987),Sanford, J. C., Trends Biotech. 6:299 (1988), Klein et al.,Bio/Technology 6:559-563 (1988), Sanford, J. C., Physiol Plant 7:206(1990), Klein et al., Biotechnology 10:268 (1992)

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol orpoly-L-ornithine have also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Saker, M.; Kuhne, T. Biologia Plantarum 40(4): 507-514(1997/98), Donn et al., In Abstracts of VIIth International Congress onPlant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin etal., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol.24:51-61 (1994). See also Chupean et al., Biotechnology. 1989, 7: 5,503-508.

Following transformation of celery target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic line. The transgenic line could then be crossed,with another (non-transformed or transformed) line, in order to producea new transgenic celery line. Alternatively, a genetic trait which hasbeen engineered into a particular celery cultivar using the foregoingtransformation techniques could be moved into another line usingtraditional backcrossing techniques that are well known in the plantbreeding arts. For example, a backcrossing approach could be used tomove an engineered trait from a public, non-elite inbred line into anelite inbred line, or from an inbred line containing a foreign gene inits genome into an inbred line or lines which do not contain that gene.As used herein, “crossing” can refer to a simple X by Y cross, or theprocess of backcrossing, depending on the context.

When the term celery plant, cultivar or celery line are used in thecontext of the present invention, this also includes any single geneconversions of that line. The term single gene converted plant as usedherein refers to those celery plants which are developed by a plantbreeding technique called backcrossing wherein essentially all of thedesired morphological and physiological characteristics of a cultivarare recovered in addition to the single gene transferred into the linevia the backcrossing technique. Backcrossing methods can be used withthe present invention to improve or introduce a characteristic into theline. The term backcrossing as used herein refers to the repeatedcrossing of a hybrid progeny back to one of the parental celery plantsfor that line. The parental celery plant which contributes the gene forthe desired characteristic is termed the nonrecurrent or donor parent.This terminology refers to the fact that the nonrecurrent parent is usedone time in the backcross protocol and therefore does not recur. Theparental celery plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman & Sleper,1994; Fehr, 1987). In a typical backcross protocol, the originalcultivar of interest (recurrent parent) is crossed to a second line(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a celeryplant is obtained wherein essentially all of the desired morphologicaland physiological characteristics of the recurrent parent are recoveredin the converted plant, in addition to the single transferred gene fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalline. To accomplish this, a single gene of the recurrent cultivar ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original line. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new line but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic, examples of these traits include but are not limited to,herbicide resistance, resistance for bacterial, fungal, or viraldisease, insect resistance, enhanced nutritional quality, industrialusage, yield stability and yield enhancement. These genes are generallyinherited through the nucleus. Several of these single gene traits aredescribed in U.S. Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, thedisclosures of which are specifically hereby incorporated by reference.

Deposit Information

A deposit of the celery cultivar seed of this invention is maintained byA. Duda & Sons, Inc. 1260 Growers Street, Salinas, California 93902,USA. Access to this deposit will be available during the pendency ofthis application to persons determined by the Commissioner of Patent andTrademarks to be entitled thereto under 37 CRF 1.14 and 35 USC 122. Uponallowance of any claims in this application, all restrictions on theavailability to the public of the variety will be irrevocably removed byaffording access to a deposit of at least 2,500 seeds of the samevariety with the American Type Culture Collection (ATCC), 10801University Boulevard, Manassas, Va. 20110.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding. However, it will be obvious that certain changes andmodifications such as single gene modifications and mutations,somaclonal variants, variant individuals selected from large populationsof the plants of the instant line and the like may be practiced withinthe scope of the invention, as limited only by the scope of the appendedclaims.

1. Seed of celery line designated ADS-3, representative seed of saidline having been deposited under ATCC Accession No. PTA-______.
 2. Acelery plant, or a part thereof, produced by growing the seed ofclaim
 1. 3. A tissue culture of regenerable cells produced from theplant of claim
 2. 4. Protoplasts produced from the tissue culture ofclaim
 3. 5. The tissue culture of claim 3, wherein cells of the tissueculture are from a tissue selected from the group consisting of leaf,pollen, embryo, root, root tip, anther, pistil, flower, seed and stem.6. A celery plant regenerated from the tissue culture of claim 3, saidplant having all the morphological and physiological characteristics ofline ADS-3, representative seed of said line having been deposited underATCC Accession No. PTA-______.
 7. A method for producing an F1 hybridcelery seed, comprising crossing the plant of claim 2 with a differentcelery plant and harvesting the resultant F1 hybrid celery seed.
 8. Ahybrid celery seed produced by the method of claim
 7. 9. A hybrid celeryplant, or parts thereof, produced by growing said hybrid seed of claim8.
 10. A method for producing a male sterile celery plant comprisingtransforming the celery plant of claim 2 with a nucleic acid moleculethat confers male sterility.
 11. A male sterile celery plant produced bythe method of claim
 10. 12. A method of producing an herbicide resistantcelery plant comprising transforming the celery plant of claim 2 with atransgene that confers herbicide resistance.
 13. An herbicide resistantcelery plant produced by the method of claim
 12. 14. The celery plant ofclaim 13, wherein the transgene confers resistance to an herbicideselected from the group consisting of: imidazolinone, sulfonylurea,glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.15. A method of producing an insect resistant celery plant comprisingtransforming the celery plant of claim 2 with a transgene that confersinsect resistance.
 16. An insect resistant celery plant produced by themethod of claim
 15. 17. The celery plant of claim 16, wherein thetransgene encodes a Bacillus thuringiensis endotoxin.
 18. A method ofproducing a disease resistant celery plant comprising transforming thecelery plant of claim 2 with a transgene that confers diseaseresistance.
 19. A disease resistant celery plant produced by the methodof claim
 18. 20. A method of producing a celery plant with modifiedfatty acid metabolism or modified carbohydrate metabolism comprisingtransforming the celery plant of claim 2 with a transgene encoding aprotein selected from the group consisting of stearyl-ACP desaturase,fructosyltransferase, levansucrase, alpha-amylase, invertase and starchbranching enzyme.
 21. A celery plant produced by the method of claim 20.22. A celery plant, or part thereof, having all the physiological andmorphological characteristics of the line ADS-3, representative seed ofsaid line having been deposited under ATCC Accession No. PTA-______. 23.A method of introducing a desired trait into celery line ADS-3comprising: (a) crossing ADS-3 plants grown from ADS-3 seed,representative seed of which has been deposited under ATCC Accession No.PTA-______, with plants of another celery line that comprise a desiredtrait to produce F1 progeny plants, wherein the desired trait isselected from the group consisting of male sterility, herbicideresistance, insect resistance and disease resistance; (b) selecting F1progeny plants that have the desired trait to produce selected F1progeny plants; (c) crossing the selected progeny plants with the ADS-3plants to produce backcross progeny plants; (d) selecting for backcrossprogeny plants that have the desired trait and physiological andmorphological characteristics of celery line ADS-3 listed in Table 1 toproduce selected backcross progeny plants; and (e) repeating steps (c)and (d) three or more times in succession to produce selected fourth orhigher backcross progeny plants that comprise the desired trait and allof the physiological and morphological characteristics of celery lineADS-3 listed in Table 1 as determined at the 5% significance level whengrown in the same environmental conditions.
 24. A plant produced by themethod of claim 23, wherein the plant has the desired trait and all ofthe physiological and morphological characteristics of celery line ADS-3listed in Table 1 as determined at the 5% significance level when grownin the same environmental conditions.
 25. The plant of claim 24 whereinthe desired trait is herbicide resistance and the resistance isconferred to an herbicide selected from the group consisting of:imidazolinone, sulfonylurea, glyphosate, glufosinate,L-phosphinothricin, triazine and benzonitrile.
 26. The plant of claim 24wherein the desired trait is insect resistance and the insect resistanceis conferred by a transgene encoding a Bacillus thuringiensis endotoxin.27. The plant of claim 24 wherein the desired trait is male sterilityand the trait is conferred by a cytoplasmic nucleic acid molecule thatconfers male sterility.